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G J Goldenberg

Publications and source records attributed to G J Goldenberg.

At least 37 records · Page 2Linked to original sources
PubMed1987

Expression of the gene encoding a prolactin-inducible protein by human breast cancers in vivo: correlation with steroid receptor status.

We have previously reported the identification and characterization of a prolactin-inducible protein (PIP) as well as the cloning of the gene encoding PIP in cultured human breast cancer cells. We now present three lines of evidence that the gene encoding PIP is also expressed by some human breast cancers in vivo: detection of PIP immunoreactivity in the serum of some breast cancer patients; immunohistochemical detection of PIP in breast cancer sections; and the presence of PIP mRNA, detected by complementary DNA hybridization in human breast biopsy samples. In a preliminary study using Western blot analysis authentic PIP was detected in the serum of some patients with breast cancer. Subsequently the sera of 234 unselected patients with breast cancer were assayed for the presence of PIP using a specific radioimmunoassay. Thirty-five % of these sera contained detectable PIP (i.e., greater than 3 ng/ml). As well as were able to show by immunohistochemical techniques that PIP immunoreactivity was present in some human breast biopsy specimens. Levels of estrogen receptor, progesterone receptor, and PIP mRNA were determined in an unselected population of 51 human breast tumor biopsies. Sixty-one % of these tumors had detectable PIP mRNA; a positive correlation (r = 0.52; P less than 0.01) was found between PIP mRNA levels in breast biopsy samples and estrogen receptor content, a known prognostic indicator in human breast cancer.

Biopsy↗
PubMed1987

Sensitivity to melphalan as a function of transport activity and proliferative rate in BALB/c 3T3 fibroblasts.

Melphalan transport by exponentially dividing BALB/c 3T3 mouse fibroblasts was approximately 3-fold greater at the steady-state than that observed with resting cells at drug concentrations ranging from 1 to 100 microM. Both the rate of incorporation of [3H]thymidine and the percentage of S-phase cells estimated by a fluorescent cell sorter provided evidence of higher DNA synthetic activity in logarithmic phase cells. Transport of melphalan by BALB/c 3T3 fibroblasts was mediated by the two amino acid transport systems, the DL-beta-2-aminobicyclo(2,2,1)heptane-2-carboxylic acid-sensitive sodium-independent system preferring leucine as substrate and the sodium-dependent system preferring alanine, serine, and cysteine as substrates. At low drug concentrations (about 1 microM) the sodium-dependent mechanism predominated, but at high concentrations of melphalan (100 microM) a shift occurred from the sodium-dependent to the sodium-independent system. Both transport systems were markedly affected by the proliferative state of the cells and became progressively less active the longer cells remained in the stationary phase. Stationary-phase cells were also less sensitive to the cytotoxic action of melphalan than exponentially dividing cells. Serum deprivation experiments showed that unidirectional melphalan influx decreased about 40% when fibroblasts previously in exponential growth were shifted from normal medium to serum-depleted medium 24 h prior to drug transport studies. Thus, serum deprivation and maintenance of cells in stationary phase growth had parallel effects on drug influx. These experiments emphasize the importance of the proliferative state of the cell as a determinant of melphalan transport and cytocidal activity.

Animals↗
PubMed1987

Modulation of membrane transport of alkylating agents and amino acids by an analog of vasopressin in murine L5178Y lymphoblasts in vitro.

The synthetic vasopressin analog 1-deamino-8-D-arginine vasopressin (dDAVP) has been shown to influence a wide range of cell-membrane-related events. Accordingly, the effect of dDAVP on membrane transport of various alkylating agents and amino acids was evaluated in L5178Y lymphoblasts in vitro. dDAVP stimulated melphalan uptake but conversely inhibited uptake of nitrogen mustard, choline (the natural transport substrate for the nitrogen mustard carrier), and leucine. No effect on the uptake of cyclophosphamide or glutamine was observed. Increased melphalan uptake was due to effects on both substrate influx and efflux. The effect of dDAVP on melphalan influx was particularly complex: dDAVP stimulated melphalan influx by amino acid transport system ASC but inhibited influx by system L, resulting in a net increase in unidirectional drug influx. Melphalan efflux was inhibited by dDAVP. Decreased uptake of nitrogen mustard, choline and leucine was due, at least in part, to decreased substrate influx. However, the mechanisms of inhibition were dissimilar: inhibition of substrate influx was non-competitive for choline but competitive for leucine. In conclusion, dDAVP induced diverse but apparently specific effects on membrane transport of several alkylating agents and amino acids. Since the accumulation of alkylating agents such as melphalan within tumor cells is a major determinant of cytotoxicity, dDAVP may have a role as a biological response modifier.

Alkylating Agents↗
PubMed1987

Transcription directed by the HIV long terminal repeat in vitro.

The long terminal repeat (LTR) of the AIDS virus (HIV) has been found to contain promoter sequences that are active in uninfected HeLa whole cell and nuclear extracts. Here we report that elements upstream of position -104 (start site +1) do not affect transcriptional activity in vitro whereas sequences between -104 and -57 are required for such activity. Using a reconstituted RNA polymerase II system, we demonstrate that a partially purified fraction containing Spl not only stimulates, as was previously reported, but is required for accurate initiation of transcription directed by the HIV LTR. In addition, based on a computerized analysis, we report the presence of a region in the HIV LTR (positions -151 to -80) that is similar to the 72 base pair enhancer element of SV40 and that includes a highly conserved segment also present in the cytomegalovirus enhancer. Moreover, the HIV and HTLV-I LTRs are shown to share a region of similarity that includes the 21 base pair motif found in the enhancers of the human and bovine T-lymphotropic viruses. The R region of the HIV LTR is found to have two extensive regions of dyad symmetry rather than one as was previously reported. The significance of these observations for HIV pathogenesis is discussed.

DNA Restriction Enzymes↗
PubMed1987

Estrogen receptor-binding affinity and cytotoxic activity of three new estrogen-nitrosourea conjugates in human breast cancer cell lines in vitro.

The hypothesis that more selective antitumor activity may be achieved by cytotoxic agents which selectively bind to estrogen receptors (ER) in human cancer cells was tested. We have synthesized three nitrosourea derivatives of estradiol or hexestrol, and compared the ER binding affinity and cytotoxic activity of these compounds against ER-positive and -negative breast cancer cell lines in vitro. Specific binding to ER in the cytosol of MCF-7 human breast cancer cells was demonstrated in these conjugates: 17 alpha-CNU greater than 17 beta-CNU greater than HEX-CNU greater than lomustine (CCNU). The order of cytotoxicity of these derivatives against human breast cancer cells appeared to correlate with their binding affinity to ER. All three estrogen nitrosourea conjugates were more cytotoxic than CCNU, a clinically useful antitumor nitrosourea which does not bind to ER. The contribution of the estrogen moiety to the cytotoxicity of 17 alpha-CNU was demonstrated by the greater activity of the conjugate than that of a combination of estrogen and CCNU. However, cytotoxicity of these compounds against the receptor-positive MCF-7 and receptor-negative Evsa-T human breast cancer cell lines was similar. The latter finding suggested that cytotoxicity of these conjugates may not be mediated through ER. The difference in stability of these nitrosourea conjugates in aqueous buffer may partly explain their differences in cytotoxicity.

Antineoplastic Agents↗
PubMed1986

Resistance to adriamycin: relationship of cytotoxicity to drug uptake and DNA single- and double-strand breakage in cloned cell lines of adriamycin-sensitive and -resistant P388 leukemia.

Cloned lines of Adriamycin (ADR)-sensitive and -resistant P388 leukemia have been established from single cell cultures. A marker chromosome M1 was found in all cells in the heterogeneous resistant P388/ADR parental line as well as in the cloned resistant lines P388/ADR/3 and P388/ADR/7; a different marker chromosome M2 was present in the heterogeneous sensitive P388 parental line as well as the cloned sensitive line P388/4. Dose-survival studies showed that D0, the dose of Adriamycin reducing survival to 1/e (i.e., 37% of the initial population), was 33 +/- 5 (SE) nM for sensitive P388/4 cells, 169 +/- 17 nM for resistant P388/ADR/3 cells, and 336 +/- 28 nM for the more resistant P388/ADR/7 cells. Drug uptake in sensitive P388/4 cells was 1.6-fold greater than in resistant P388/ADR/3 cells and 2.1-fold greater than in resistant P388/ADR/7 cells. The number of DNA single-strand breaks produced per microM Adriamycin was 131 +/- 9 rad equivalents in sensitive clone 4 cells, 41 +/- 8 rad equivalents in resistant clone 3 cells, and 33 +/- 11 rad equivalents in resistant clone 7 cells. The number of DNA double-strand breaks per microM Adriamycin was 1721 +/- 126 rad equivalents in sensitive cells, 117 +/- 36 rad equivalents in resistant P388/ADR/3 cells, and 194 +/- 16 rad equivalents in resistant P388/ADR/7 cells. Differences in drug uptake were insufficient to explain the higher incidence of DNA single- and double-strand breaks in sensitive cells. These findings strongly support the concept that resistance to Adriamycin in P388 leukemia cells is multifactorial; however, this study did not resolve whether these changes arise from a single pleiotropic mutation or from multiple mutations. In sensitive P388/4 cells the number of DNA single-strand breaks formed could all be attributed to double-strand breaks. However, in both resistant cell lines the level of induction of single-strand breaks was in excess of that due to double-strand breaks, and this excess of single-strand breaks appeared to vary directly with the degree of resistance, being greater in the more resistant clone 7 cells than in the less resistant clone 3 cells. In both sensitive and resistant cell lines the ratio of true single- to double-strand breaks varied inversely with the concentration of Adriamycin. Finally, the cytotoxic activity of Adriamycin appeared to correlate more closely with formation of DNA double-strand breaks than with single-strand lesions.

Animals↗
PubMed1986

Comparison of adriamycin uptake in chick embryo heart and liver cells an murine L5178Y lymphoblasts in vitro: role of drug uptake in cardiotoxicity.

The mechanism of uptake of Adriamycin was investigated in chick embryo heart and liver cells and in murine L5178Y lymphoblasts in vitro. Drug uptake at 4 degrees C for 15 s due to rapid association accounted for a cell:medium distribution ratio of 104 +/- 14 (SE) in heart cells, 10.2 +/- 1.3 in liver cells, and 10.3 +/- 1.5 in L5178Y lymphoblasts. On thin-layer chromatographic analysis, 98% of the radioactivity migrated with the mobility of intact drug, suggesting that drug metabolism was negligible for at least 30 min in both heart and tumor cells. A time course of drug uptake was somewhat different in heart cells compared to that noted for liver or L5178Y cells. The steady state for drug uptake was reached more promptly in heart cells; apparent equilibrium was observed at 6 min in heart cells, at approximately 20 min in L5178Y lymphoblasts, but was not attained by 25 min in liver cells. Temperature dependence of drug uptake also differed in the three cell types; drug uptake was most temperature sensitive in L5178Y cells, intermediate in liver cells, and least temperature dependent in heart cells. Separation of heart, liver, and leukemic cells into membrane and cytosol fractions demonstrated that, at 1 and 30 min, more than 75% of the drug was associated with the membrane fraction. Trichloroacetic acid extraction of cell constituents revealed that, at 1 min, the acid-soluble fraction amounted to 32 +/- 2% of radioactivity in heart cells and 37 +/- 2% in L5178Y cells. Ethanol extraction of these cells demonstrated that, at 1 min, ethanol-soluble components accounted for 49 +/- 2% of radioactivity in heart cells and 27 +/- 2% in leukemic cells. The finding of a large component of rapid association together with evidence of prompt drug binding to cellular constituents made evaluation of unidirectional drug influx impractical. Accordingly, an investigation was undertaken of Adriamycin efflux from chick embryo heart and liver cells and L5178Y lymphoblasts, after the cells had been loaded with drug for various time intervals. In all three cell types, efflux was rapid down to a plateau level, representing nonexchangeable drug. As the period of time for loading cells was increased, there was a progressive rise in the level of nonexchangeable drug. Equilibration of the nonexchangeable pool occurred more rapidly in heart cells than in either liver or leukemic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Antagonism of the cytocidal activity and uptake of melphalan by tamoxifen in human breast cancer cells in vitro.

The effect of the antiestrogen tamoxifen on the cytocidal activity and uptake of melphalan in human breast cancer cells was investigated. A clonogenic assay was used to obtain dose-survival curves of estrogen receptor-positive MCF-7 cells and of estrogen receptor-negative Evsa T cells following treatment with melphalan and/or tamoxifen. Isobolograms derived from these dose-survival curves were concave downward, suggesting that the drug interaction was antagonistic. The effect of tamoxifen on melphalan uptake by breast cancer cells was evaluated at steady-state conditions. Thin-layer chromatography revealed that the intracellular level of free intact melphalan (mean +/- S.E.) in control cells was 6.47 +/- 1.21 fmoles/cell and that in cells treated with tamoxifen was 3.60 +/- 0.35 fmoles/cell; this 44% reduction in cellular melphalan was statistically significant (P = 0.006). Thus, the antagonistic cytocidal effect of melphalan and tamoxifen against breast cancer cells appeared to be due to inhibition of melphalan uptake at the steady state by the antiestrogen. Further investigation revealed that tamoxifen inhibited unidirectional melphalan influx in human breast cancer cells both by the sodium-independent system L and by the sodium-dependent system ASC. Tamoxifen also appeared to stimulate melphalan efflux from human breast cancer cells. The first-order rate constant K for melphalan efflux from control cells was 0.085 +/- 0.008 and that from cells treated with tamoxifen was 0.129 +/- 0.005; the difference was highly significant (P less than 0.001). Therefore, the antagonistic effect of tamoxifen on the uptake and cytocidal activity of melphalan in breast cancer cells appeared to be due to inhibition of melphalan influx and stimulation of drug efflux.

Breast Neoplasms↗
PubMed1985

Induction of DNA single- and double-strand breaks by diethylstilbestrol in murine L5178Y lymphoblasts in vitro.

The mechanism of action of the synthetic estrogen diethylstilbestrol (DES) was investigated in murine L5178Y lymphoblasts. The dose-survival curve of cells treated with DES in serum-free medium for 1 hr was characterized by a prominent shoulder followed by a simple exponential decline; the Do, the dose of DES reducing cell survival to 1/e, was 1.52 nmoles/ml. DNA single-strand breaks, as measured by the alkaline elution method, were observed in DES-treated cells, and these followed a dose-response relationship after an apparent threshold of 10 microM DES was exceeded. Protein-associated strand breaks, which represent the increment in single-strand breaks that occurs by exposing drug-treated cells to proteinase K, were also noted. DNA double-strand breaks as measured by filter elution technology at pH 9.6 were observed and increased markedly to reach a level of approximately 9000 rad equivalents at a DES concentration of 20 microM. The measured ratio (mean +/- S.E.) of single- to double-strand breaks induced by DES in L5178Y limphoblasts was 0.09 +/- 0.035. A comparison of the ratio of single- to double-strand breaks induced by DES to that observed following radiation suggested that all of the single-strand breaks produced by DES could be attributed to double-strand breaks. The close correspondence of the dose-response curve for cytocidal activity of DES with that obtained for induction of DNA double-strand breaks suggested that such breaks may play an important role in the mechanism of cell kill by DES.

Animals↗
PubMed1983

Uptake and decomposition of chlorambucil by L5178Y lymphoblasts in vitro.

The uptake of [14C]chlorambucil by L5178Y lymphoblasts was studied using thin-layer chromatography to identify the various radioactive components that enter or leave cells. Theoretical calculations predicted that entry of chlorambucil into cells by simple diffusion would be rapid and essentially complete in 45 sec or less. Uptake of intact chlorambucil was rapid, reaching a cell/medium ratio of approximately 1.5 in less than 15 sec at both 37 degrees and 4 degrees, consistent with a simple diffusion mechanism. In cells treated with [14C]chlorambucil for 60 min, the intracellular level of intact drug decreased with time, and this decay was attributed to hydrolysis and alkylation. The level of intact drug in the medium decreased at a similar rate resulting in a nearly constant cell/medium distribution ratio. Intact chlorambucil in the cells was found to be entirely ethanol- and trichloroacetic acid-soluble. Efflux of intact chlorambucil was very rapid and temperature-insensitive. These findings suggest that chlorambucil efflux, as well as influx, is by a simple diffusion mechanism. A derivative of chlorambucil was found in ethanol solutions of the drug. This derivative, which may be the ethyl ester of the drug, is highly concentrated in cells and may interfere with pharmacological investigations of chlorambucil.

Alkylation↗
PubMed1982

Mechanism of efflux of melphalan from L5178Y lymphoblasts in vitro.

The mechanism of efflux of melphalan from L5178Y lymphoblasts in vitro was investigated. Evidence was obtained that drug efflux occurs by a process different from the influx mechanism. A time course of efflux followed a first-order process for at least 5 min, with a rate constant K of 0.13 +/- 0.05 (S.D.) min-1, and approximately 80% of drug taken up by the cells was exchangeable. From a kinetic analysis of melphalan efflux, it was not possible to determine if the mechanism of drug efflux was simple diffusion or a technically nonsaturable carrier-mediated process. The Q10 for drug efflux varied from 1.2 to 1.8, values intermediate between those expected for simple diffusion and a carrier-mediated process. Furthermore, drug efflux was sodium independent and was not inhibited by the presence of amino acids on the same side of the membrane, findings which support the concept that efflux was by simple diffusion. The presence of a wide variety of amino acids in the extracellular medium was found to stimulate melphalan efflux. An evaluation of the structure-activity relationship disclosed that amino acids containing hydroxyl, acidic, or amide side chains were most active; however, a distinct pattern between amino acid structure and stimulation of efflux was not established. The stimulation process was concentration dependent and was not restricted to either L5178Y cells or to melphalan as transport substrate.

Amino Acids↗
PubMed1982

Drug and hormone sensitivity of estrogen receptor-positive and -negative human breast cancer cells in vitro.

A clonogenic assay of long-term breast cancer cell cultures in vitro has been developed to provide a highly reproducible method with which to quantitate tumor cell killing by hormones and/or cytotoxic chemotherapeutic agents. Monolayer cultures of estrogen receptor-positive MCF-7 human breast cancer cells and of estrogen receptor-negative Evsa T cells are harvested by treatment with 0.01% trypsin:0.02% EDTA in Hanks' balanced salt solution. Cell suspensions are treated with drug or hormone in serum-free medium for 1 hr at 37 degrees; treated cells are washed, plated, and cultured for approximately 14 days; and colonies consisting of greater than or equal to 30 cells are counted. Compared to estrogen receptor-positive cells, estrogen receptor-negative cells were 2-fold more sensitive to melphalan but were conversely 1.9-fold more resistant to Adriamycin; these differences were statistically significant (p less than 0.001). Thus, response to cytotoxic chemotherapeutic agents appeared to be independent of estrogen receptor status. For cells treated with diethylstilbestrol, the dose of drug or hormone reducing the surviving cell fraction to 1/e (DO) for estrogen receptor-positive cells was 2.27 nmol/ml, and that for estrogen receptor-negative cells was 2.80 nmol/ml; this difference was not statistically significant. However, with tamoxifen therapy, the DO for estrogen receptor-positive cells was 0.601 nmol/ml, and that for estrogen receptor-negative cells was 3.64 nmol/ml; this 6-fold greater degree of resistance to tamoxifen of estrogen receptor-negative cells was highly significant (p less than 0.001). Treatment of cells for 24 hr with 17 beta-estradiol stimulated proliferation not only of estrogen receptor-positive cells but also of estrogen receptor-negative cells. However, estradiol at concentrations up to 200 microM had no apparent cytocidal activity, as measured by the clonogenic assay. Furthermore, treatment of MCF-7 cells simultaneously with estradiol and either diethylstilbestrol or tamoxifen failed to reverse the cytocidal activity of those two agents. These findings suggest that, in the clonogenic assay described herein, diethylstilbestrol and tamoxifen may kill human breast cancer cells by an independent mechanism of action and that the cytocidal activity of diethylstilbestrol and the proliferative effect of 17 beta-estradiol appear to be independent of estrogen receptor status.

Breast Neoplasms↗
PubMed1980

A comparison of melphalan transport in human breast cancer cells and lymphocytes in vitro.

Evidence is presented that melphalan uptake by MCF-7 human breast cancer cells and peripheral blood lymphocytes from normal human subjects is an active process involving 2 amino acid carriers. Quantitative differences were observed in drug transport between the 2 cell types. Cell/medium distribution ratios of melphalan at equilibrium conditions were approx. 4-fold higher in breast cancer cells than in lymphocytes. The transport capacity, Vmax, for both carrier systems was at least 50-fold greater in MCF-7 cells than in lymphocytes, whereas the Michaelis constants were similar. These findings, in part, provide a rational basis for the use of melphalan in the chemotherapy of breast cancer.

Amino Acids↗
PubMed1980

Uptake and decomposition of chlorozotocin in L5178Y lymphoblasts in vitro.

Uptake and metabolism of 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin) by L5178Y lymphoblasts in vitro was investigated, using both glucose- and chloroethyl-labeled chlorozotocin. A time course of uptake of total radioactivity revealed a greater cell/medium distribution ratio of activity in cells treated with chloroethyl-labeled chlorozotocin compared to cells treated with the glucose-labeled compound. Thin-layer chromatographic analysis showed that uptake of intact chlorozotocin was identical in cells treated with either glucose- or chloroethyl-labeled drug and that the cell/medium distribution ratio never exceeded unity. Accumulation of 14C-chlorozotocin was not inhibited by an excess of unlabeled chlorozotocin, the structural analogs glucose and glucosamine, or several metabolic inhibitors or by sodium ion depletion. These observations, together with the relatively low temperature quotient for the uptake process, suggested that chlorozotocin uptake occurs by passive diffusion. In cells treated with glucose-labeled chlorozotocin, a bicyclic urethan derivative and polar metabolites soluble in trichloroacetic acid were formed. In cells exposed to chloroethyl-labeled drug, nonpolar as well as polar metabolites were noted. Formation of metabolites from the glucose moiety was impeded by the presence of an excess of unlabeled chlorozotocin, the structural analogs glucose and glucosamine, the glucose transport inhibitors phlorizin and phloretin, the metabolic inhibitor m-chlorophenyl carbonyl cyanide hydrazone and by sodium depletion. Appearance of metabolites arising from the chloroethyl moiety was also blocked by the presence of m-chlorophenyl carbonyl cyanide hydrazone and by sodium ion depletion. These results suggested that metabolism of chlorozotocin in L51789Y lymphoblasts appears to be enzyme mediated.

Animals↗
PubMed1979

Active carrier-mediated transport of melphalan by two separate amino acid transport systems in LPC-1 plasmacytoma cells in vitro.

Previous studies have shown that uptake of several alkylating agents occurs by independent transport mechanisms. Uptake of one of these agents, the phenylalanine derivative of nitrogen mustard (melphalan), has been investigated in LPC-1 plasmacytoma cells in vitro. Evidence suggesting that melphalan uptake is an active process is that uptake of free intact melphalan proceeds "uphill" against a concentration gradient, is temperature- and sodium-dependent, and is inhibited by several metabolic antagonists. Other findings supporting the concept that melphalan uptake is carrier-mediated is that uptake follows biphasic Michaelis-Menten kinetics, is chemically specific, and is mediated by at least two separate amino acid transport systems. Uptake by one system was sensitive to DL-beta-2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid, was sodium-independent, and appeared to be mediated by system L. The second unassigned system was characterized by sodium dependence, insensitivity to 2-aminoisobutyric acid and 2-(methylamino)isobutyric acid, and was inhibited by alanine, serine, cysteine, and other amino acids.

Amino Acids↗